Various processes have been employed for the removal of nicotine from tobacco. Most of those processes, however, are not sufficiently selective for nicotine. They remove other ingredients from the tobacco, thereby adversely affecting its flavor and aroma. In addition, such processes are typically complex and expensive.
Nicotine, and biologically synthesized compounds in general, are formed through sequences of biochemical reactions, wherein each reaction is catalyzed by a different enzyme. The particular reaction sequence leading to a given compound is known as a pathway. One approach for inhibiting the operation of a pathway, and thus output of its end product, is reducing the amount of a required enzyme in the pathway. If the enzyme's abundance, relative to the other enzymes of the pathway, is normally low enough to make that enzyme rate-limiting in the pathway's operation, then any reduction in the enzyme's abundance will be reflected in lowered production of the end product. If the enzyme's relative abundance is not normally rate limiting, its abundance in the cell would have to be reduced sufficiently to make it rate-limiting, in order for the pathway's output to be diminished. Similarly, if the enzyme's relative abundance is rate limiting, then any increase in its abundance will result in increased production of the pathway's end product.
Nicotine is formed primarily in the roots of the tobacco plant and subsequently is transported to the leaves, where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden, Hutchinson & Ross, Stroudsburg, PA (1972)). The nicotine molecule is comprised of two heterocyclic rings, a pyridine moiety and a pyrrolidine moiety, each of which is derived from a separate biochemical pathway. The pyridine moiety of nicotine is derived from nicotinic acid. The pyrrolidine moiety of nicotine is provided through a pathway leading from putrescine to N-methylputrescine and then to N-methylpyrroline. An obligatory step in nicotine biosynthesis is the formation of N-methylputrescine from putrescine (Goodwin and Mercer, Introduction to Plant Biochemistry, pp. 488-91, Pergamon Press, New York, (1983)).
Conversion of putrescine to N-methylputrescine is catalyzed by the enzyme putrescine N-methyltransferase ("PMT"), with S-adenosylmethionine serving as the methyl group donor. PMT appears to be the rate-limiting enzyme in the pathway supplying N-methylpyrroline for nicotine synthesis in tobacco (Feth et al., "Regulation in Tobacco Callus of Enzyme Activities of the Nicotine Pathway", Planta, 168, pp. 402-07 (1986); Wagner et al., "The Regulation of Enzyme Activities of the Nicotine Pathway in Tobacco", Physiol. Plant., 68, pp. 667-72 (1986)).
A relatively crude preparation of PMT (30-fold purification) has been subjected to limited characterization (Mizusaki et al., "Phytochemical Studies on Tobacco Alkaloids XIV. The Occurrence and Properties of Putrescine N-Methyltransferase in Tobacco Plants", Plant Cell Physiol., 12, pp. 633-40 (1971)). The purification steps leading to that preparation were limited to ammonium sulfate precipitation from the initial extract and gel filtration chromatography. Id.
Antisense RNA technology allows the production of plants characterized by levels of an enzyme (or other protein) that are significantly lower than those normally contained by the plants. Ordinarily, transcription of a gene coding for a target enzyme gives rise to a single-stranded mRNA, which is then translated by ribosomes to yield the target enzyme. An antisense RNA molecule is one whose nucleotide sequence is complementary to some portion of the target mRNA molecule. The antisense RNA molecule, thus, will undergo complementary base pairing (hybridization) with the target mRNA molecule, rendering the target mRNA molecule unavailable for translation, more susceptible to degradation, or both. The ability of the cell to produce the specific enzyme coded for by the target mRNA is thus inhibited.
Antisense technology has been employed in several laboratories to create transgenic plants characterized by lower than normal amounts of specific enzymes. For example, plants with lowered levels of chalcone synthase, an enzyme of a flower pigment biosynthetic pathway, have been produced by inserting a chalcone synthase antisense gene into the genome of tobacco and petunia. These transgenic tobacco and petunia plants produce flowers with lighter than normal coloration (Van der Krol et al., "An Anti-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation", Nature, 333, pp. 866-69 (1988)). Antisense RNA technology has also been successfully employed to inhibit production of the enzyme polygalacturonase in tomatoes (Smith et al., "Antisense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes", Nature, 334, pp. 724-26 (1988); Sheehy et al., "Reduction of Polygalacturonase Activity in Tomato Fruit by Antisense RNA", Proc. Natl. Acad. Sci. USA, 85, pp. 8805-09 (1988)), and the small subunit of the enzyme ribulose bisphosphate carboxylase in tobacco (Rodermel et al., "Nuclear-Organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants", Cell, 55, pp. 673-81 (1988)). Alternatively, transgenic plants characterized by greater than normal amounts of a given enzyme may be created by transforming the plants with the gene for that enzyme in the sense (i.e., normal) orientation.
Genetic engineering of tobacco plants to lower or raise levels of nicotine through altered levels of PMT has not been possible, because a method for cloning PMT genes without prior purification of the PMT enzyme is not known, and a method for purification of the PMT enzyme was not known prior to this invention.